Method for micropropagation of monocots based on sustained totipotent cell cultures

ABSTRACT

The present invention provides a method of micropropagating a monocotyledonous plant comprising: (a) cultivating an explant of tissue from a monocotyledonous plant shoot tip on a primary medium, wherein the explant has been pretreated with a cold temperature and the primary medium comprises auxin or auxin and cytokinin, to produce a totipotent embryogenic cell culture; (b) treating the totipotent embryonic cell culture with a cold temperature; (c) maintaining the totipotent embryogenic cell culture by cultivation on a secondary medium, whereby a totipotent embryogenic cell culture of a monocotyledonous plant is produced and maintained; and (d) transferring the embryogenic cell culture of step (c) to a tertiary medium to continue multiplication and to produce a plantlet with roots and shoots, thereby micropropagating a monocotyledonous plant. The micropropagation techniques described herein provide plants for such purposes as development of elite plant lines, phytoremediation and biomass production.

STATEMENT OF GOVERNMENT SUPPORT

The present invention was funded in part by government support undergrant number 10049180 from the National Institutes of Health. The UnitedStates Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to novel methods for large-scalemicropropagation of plants of the Class Monocotyledoneae.

BACKGROUND OF THE INVENTION

Plant regeneration from cultured cells of the great majority of monocot(mostly graminaceous) species that have been reported so far, isachieved from callus initiated on high concentrations of a strong auxin,such as 2,4-dichlorophenoxyacetic acid (2,4-D) (Conger, B. V. et al.,Current Issues in Plant Molecular and Cellular Biology, pp. 59-68(1995)). The synthetic auxin, 2,4-D, is considered to be the best planthormone to induce embryogenic callus. Embryogenic callus is typicallyobtained from monocots by inducing the primary cell culture on a mediumcontaining one or more auxin-type plant hormones followed by a secondarycultivation step on a lowered auxin but in the presence of acytokinin-type plant hormone. The embryogenic potential of monocot cellcultures diminishes over time, making it necessary to reinitiate theprimary cell culture (U.S. Pat. No. 6,153,812 issued Nov. 28, 2000;Trigiano and Gray, Plant tissue culture concepts and laboratoryexercises. Second ed., Boca Raton: CRC Press (2000)).

Plants from the Class Monocotyledoneae are often multipurpose plants.Giant reed, Arundo donax, for example, has been used for 5,000 years forpipe instruments and is the source for reeds for clarinets and organpipes. Even with today's modern technology, most of the reeds forwoodwind musical instruments are still made from A. donax culms.

Giant reed is also used for erosion control and has great potential foruse as an energy crop (Szabo, P., et al., J. Anal. Appl. Pyrolysis,36:179-190 (1996)). The culms are also used for fishing rods, walkingsticks, mats and lattices in the construction of adobe huts. Giant reedis also a source of industrial cellulose for paper and rayon making, andfor the production of other polysaccharides (Neto, C. P. e al., Ind.Crops & Prods., 6:51-58 (1997). It has even been considered as a sourceof pulp for the making of paper. Additionally, giant reed can also beused in bioremediation efforts to remove environmental pollutants fromwater and land areas.

Giant reed is only one of many monocots that exhibit such multiple uses.Whether used as ornamentals, sources of energy, or as useful vehicles tocarry out industrial processes, such grass-like plants are important.

The present inventors have found that 2,4-D induced callus culturesobtained from giant reed nodal segments and segments of immatureinflorescences fail to produce sustained embryogenic cell cultures andembryos even upon transfer to low auxin or no auxin in combination witha cytokinin, as is the typical practice (U.S. Pat. No. 6,153,812 issuedNov. 28, 2000; Trigiano and Gray, Plant tissue culture concepts andlaboratory exercises. Second ed., Boca Raton: CRC Press (2000)). Thefinding that these methods fail to produce embryogenic cultures iscorroborated by Tóth and Mix-Wagner and Linder and Gallagher where Tóthand Mix-Wagner reported the formation of callus that was claimed to beembryogenic but they failed to obtain embryos and plant regeneration andLinder and Gallagher announced one-time plant regeneration from calluswithout the potential for mass propagation (Tóth and Mix-Wagner,Sustainable agriculture for food, energy and industry: strategiestowards achievement: proceedings of the international conference held inBraunschweig, Germany, June 1997, N. El Bassarn, et al., Eds., James &James (Science Publishers) Ltd.: London. pp. 249-253 (1998); Linder andGallagher, Abstract No. 257, American Journal of Botany, 85(6): 89(1998)).

Accordingly, it would be useful to have methods for the large-scalemicropropagation and macropropagation of Arundo donax and other monocotplants. Thus, the present invention provides a comprehensive set ofmethods for the production, propagation, maintenance, storage,transportation and deployment of sustained embryogenic cell cultures aswell as derived secondary and tertiary totipotent tissue cultures fromplant species of the Class Monocotyledoneae and from elite plant linesderived therefrom. The plant micropropagation technology of the presentinvention based on the sustained embryogenic cultures providesunprecedented efficiency.

SUMMARY OF THE INVENTION

The present invention provides a comprehensive set of methods for theinitiation, mass micropropagation, maintenance, storage, transportation,and/or for deployment of propagules. The present invention furtherprovides methods for enhanced somaclonal breeding of monocotyledousplant species utilizing the sustained embryogenic cell culture inductionand maintenance technology described herein. The methods of the presentinvention can thus provide superior plant clones in commercially viablenumbers at an industrial scale that can be used for many purposesincluding remediation and biomass plantations. Thus, the presentinvention further provides methods for industrial scale, cost effective,mass production of field ready propagules for the production of monocotplants using multiple or a single subculture technology from embryogeniccell cultures.

Accordingly, one embodiment of the present invention is a method ofproducing and maintaining a totipotent embryogenic cell culture of amonocotyledonous plant comprising: (a) cultivating an explant of tissuefrom a monocotyledonous plant shoot tip on a primary medium, wherein theexplant has been pretreated with a cold temperature and the primarymedium comprises auxin or auxin and cytokinin, to produce a totipotentembryogenic cell culture; (b) treating the totipotent embryonic cellculture with a cold temperature; and c) maintaining the totipotentembryogenic cell culture by further cultivation on the primary mediumand/or on a secondary medium, whereby a totipotent embryogenic cellculture of a monocotyledonous plant is produced and maintained.

A further embodiment provides a method of micropropagating amonocotyledonous plant comprising: (a) cultivating an explant of tissuefrom a monocotyledonous plant shoot tip on a primary medium, wherein theexplant has been pretreated with a cold temperature and the primarymedium comprises auxin or auxin and cytokinin, to produce a totipotentembryogenic cell culture; (b) treating the totipotent embryonic cellculture with a cold temperature; (c) maintaining the totipotentembryogenic cell culture by cultivation on a secondary medium, whereby atotipotent embryogenic cell culture of a monocotyledonous plant isproduced and maintained; and (d) transferring the embryogenic cellculture of step (c) to a tertiary medium to continue multiplication andto produce a plantlet with roots and shoots, thereby micropropagating amonocotyledonous plant.

In still further embodiments, a method of micropropagating amonocotyledonous plant is provided comprising: (a) cultivating anexplant of tissue from a monocotyledonous plant shoot tip on a primarymedium, wherein the explant has been pretreated with a cold temperatureand the primary medium comprises auxin or auxin and cytokinin, toproduce a totipotent embryogenic cell culture; (b) treating thetotipotent embryonic cell culture with a cold temperature; (c)maintaining the totipotent embryogenic cell culture by cultivation on asecondary medium, whereby a totipotent embryogenic cell culture of amonocotyledonous plant is produced and maintained; (d) transferring theembryogenic cell culture of step (c) to a tertiary medium to continuemultiplication and to produce a plantlet with roots and shoots; and (e)transferring the plantlet to a quaternary medium for acclimatization tonon-sterile and photosynthetic conditions and production of anacclimatized plantlet or plant.

Another embodiment of the present invention provides a method ofproducing an elite plant line comprising: selecting for at least onetrait of interest in the totipotent embryogenic cell culture and/orplantlet produced by the methods of the present invention, wherein theat least one trait of interest is a result of somaclonal variation inthe embryogenic cell culture or the introduction of at least oneheterologous nucleotide sequence into the genome of a cell of theembryogenic cell culture; and cultivating the totipotent embryogeniccell culture comprising the at least one trait of interest to produce anelite plant line.

A further embodiment provides a method wherein a plant-microbeassociation is established, the method comprising co-cultivating atleast one plantlet produced by the methods of the present invention withat least one microbial species in a quaternary medium to establish aplant-microbe association.

The present invention additionally provides methods directed tophytoremediation. In one embodiment, the phytoremediation comprisesestablishing a plurality of plants produced by methods of the presentinvention and possessing the same genetic characteristics in a liquidmedium, and contacting the roots of the plants with an environmentalpollutant in the liquid medium, thereby causing the environmentalpollutant to be removed from the liquid medium.

In other embodiments, phytoremediation comprises establishing aplurality of plants produced by methods of the present invention andpossessing the same genetic characteristics in a land area andcontacting the roots of the plants with an environmental pollutant inthe land area, thereby causing the environmental pollutant to be removedfrom the land area.

Still further embodiments of the present invention include a totipotenttissue or a transgenic totipotent tissue of a monocot plant that isproduced by the methods described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows sustained totipotent embryogenic cell culture of Arundodonax. FIG. 1A shows the first step in embryogenesis on secondaryculture medium, asymmetric cell division; FIG. 1B shows the highlycytoplasmic, actively dividing cells in embryogenic cell culture onsecondary culture medium; and FIGS. 1C and 1D show nonzygotic embryofrom cell culture on secondary culture medium.

FIG. 2 shows the stages of industrial scale propagation. Step 1 is theinitiation of the embryogenic cell culture on primary cultivation medium(I). The embryogenic cell culture can be propagated on primary culturemedium with or without intermittent cold treatment. Step 2 is thetransfer to secondary culture medium (II) where the cell culture can bepropagated with or without intermittent cold-treatment. Step 3 shows thetransfer from secondary to tertiary culture medium (III). Step 4illustrates an alternative route wherein the cell culture from theprimary cultivation medium is transferred directly to the quaternarycultivation medium (IV). Step 5 is acclimatization of the cultures onthe quaternary cultivation medium. Step 6 is the transfer of the plantsinto soil.

FIG. 3 shows the stages of in vitro culture, multiplication, andregeneration of Arundo donax via sustained totipotent embryogenic cellculture. FIG. 3A shows the formation of embryogenic cell culture onimmature inflorescence segments; FIG. 3B shows sustained totipotentembryogenic cell culture on secondary culture medium; FIG. 3C showsunipolar nonzygotic embryos forming on the sustained totipotentembryogenic cell culture on secondary culture medium; FIG. 3D showsplants regenerated from the embryogenic cell culture and multiplying inliquid tertiary culture medium; FIG. 3E shows plants transferred to potsfrom the quaternary culture medium; and FIG. 3F shows a three-month-oldstand of A. donax established from in vitro cloned plants.

FIG. 4 shows the stages of in vitro culture, multiplication, andregeneration of Miscanthus×giganteus via sustained totipotentembryogenic cell culture. FIGS. 4A and 4B show sustained totipotentembryogenic cell culture on secondary culture medium; FIGS. 4C and 4Dshow plants regenerating from the embryogenic cell culture on tertiaryculture medium in the dark; and FIG. 4E shows plants in liquid tertiaryculture medium under light.

FIG. 5 provides examples of different monocot species produced using themethods described herein. FIG. 5A. Miscanthus floridulus in thesecondary cultivation stage; FIG. 5B. Thysanolaena maxima in thesecondary cultivation stage; FIG. 5C Sciipus validus in theacclimatization stage; FIG. 5D Scirpus californicus in the tertiarycultivation stage.

FIG. 6 shows selection of antibiotic resistant transformants four weeksafter Agrobacterium-mediated gene transfer to cross-sectional segmentsof immature Arundo donax inflorescence (explants). Transformation iscarried out by cocultivation of the embryogenic callus withAgrobacterium tumefaciens carrying plasmid pMSF3022. FIG. 6A showscontrol explants killed by 10 mg/L of phosphinothricin(antibiotic/herbicide); FIG. 6B shows the development of embryogeniccallus in the absence of the antibiotic/herbicide in the case of thecontrol explants; FIG. 6C shows that co-cultivated antibiotic resistanttransformed explants are able to develop embryogenic callus in thepresence of 10 mg/L of phosphinothricin; and FIG. 6D shows thedevelopment of embryogenic callus in the case of cocultivated antibioticresistant transformed explants in the absence of theantibiotic/herbicide.

FIG. 7 shows the differences in dispersion of the genetic variability inthe dehalogenase activity on the halogenated xenobiotic,2,4,6-trichlorophenol (TCP). FIG. 7A shows the dispersion of vegetativeclones, obtained by conventional nursery propagation, with respect todehalogenase activity; FIG. 7B shows the dispersion of somaclonesobtained from embryogenic cell cultures; FIG. 7C shows the dispersion ofsomaclones obtained from embryogenic cell cultures exposed to TCP; andFIG. 7D presents the data charted as quartiles.

FIG. 8 shows an example of a microfilm degradation assay. Arundo donaxroots colonized by bacteria (Pseudomonas cepaica) show clearing aroundthe roots where the oil film is digested.

DETAILED DESCRIPTION

The present invention will now be described more fully hereinafter withreference to the accompanying drawings and specification, in whichpreferred embodiments of the invention are shown. This invention may,however, be embodied in different forms and should not be construed aslimited to the embodiments set forth herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. As used in the description of the invention and the claimsset forth herein, the singular forms “a,” “an,” and “the” are intendedto include the plural forms as well, unless the context clearlyindicates otherwise. As used herein, the term “and/or” includes any andall combinations of one or more of the associated listed items.

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

All publications, patent applications, patents and other referencescited herein are incorporated by reference in their entireties for theteachings relevant to the sentence and/or paragraph in which thereference is presented.

DEFINITIONS

As used herein “auxin” refers to any compound with auxin or auxin-likeactivity. “Auxin-like activity” refers to the typical activity observedin a plant as a result of treatment with auxin. Thus, a compound withauxin-like activity promotes the formation of unorganized cell mass onexplants, maintains cell proliferation alone or in the presence ofcytokinin and/or induces root development on shoot cuttings.

As used herein “cold pretreatment” refers to the cold treatment ofexplants prior to introduction into cell culture.

As used herein “cold treatment” refers to incubation of the establishedembryogenic cell cultures in the cold.

As used herein “cytokinin” refers to any compound with cytokinin orcytokinin-like activity. “Cytokinin-like activity” refers to the typicalactivity observed in a plant as a result of treatment with cytokinin.Thus, a compound with cytokinin-like activity promotes shootregeneration from cell cultures and/or maintains cell proliferation inthe presence of auxin.

Embryo is defined as the earliest recognizable multicellular stage of anindividual.

“Embryogenic cultures” as used herein refers to cultures that canproduce somatic embryos or embryo derived structures, which are able tofurther differentiate and form a plantlet.

As used herein, the phrase “increased tolerance to an environmentalcondition” means the ability of a plant to withstand a deleteriousenvironmental condition that would normally be harmful or deleterious toa wild-type plant of the same species.

“Plantlet” as used herein refers to a young or small plant with rootsand shoots. In some embodiments of the present invention, the plantletsare 0.5-10 cm in height.

“Primary totipotent embryogenic cell culture” as used herein refers to anon-organized mass of actively dividing undifferentiated cells which areable to differentiate into nonzygotic embryogenic structures, embryosand embryo derived structures which can further differentiate and form aplantlets.

“Propagule” as used herein refers to plants and plantlets that are usedfor propagating a plant and are ready to be transplanted into soil orother planting medium.

“Rehabilitate” or “rehabilitation” and other like terms as used hereinrefer to transferring of the cultures, which have been in storage, tofresh medium.

As used herein, the term “subculture,” or “passage” refers to thetransfer of cells from one culture vessel to another; this usuallyinvolves the subdivision of a proliferating cell culture. Thus,“subculture” is the process by which the tissue or explant is subdividedand then transferred into fresh culture medium.

“Sustained totipotent embryogenic cell culture” as used herein refers toa mixture of non-organized actively dividing undifferentiated cells andcells differentiating into nonzygotic embryogenic structures, embryosand embryo derived structures, which can further differentiate and formplantlets for years.

As used herein “totipotent” means having unlimited capability to produceany type of cell. Totipotent cells have the capability to turn (or“specialize”) into any and all of the tissues and organs that arepresent in the completely developed plant. Thus, totipotent cells havethe capability to regenerate into whole plants.

As used herein “xenobiotic” refers to a chemical or other compound whichis not a normal component of an organism which is exposed to it; achemical that is foreign to the biological system.

It will be understood that, although the terms “primary”, “secondary”,etc. may be used herein to describe various cultivation steps and/ormedia, these cultivation steps and/or media should not be limited bythese terms. These terms are only used to distinguish one cultivationstep or media from another cultivation step or media. Thus, a “primary”cultivation step or media, discussed below could also be termed a“secondary” cultivation step or media without departing from theteachings of the present invention.

The present invention provides new methods for the initiation, massmicropropagation, maintenance, storage, transportation and/or deploymentof propagules as well as for enhanced somaclonal breeding of selectplant species. In representative embodiments, the present inventionfurther provides plant clones in commercially viable numbers at anindustrial scale for among other uses, remediation and biomassplantations. These methods utilize a sustained embryogenic cell cultureinduction and maintenance technology that to the inventors' knowledgeprovides the first sustained embryogenic cell culture of such monocotsas giant reed, tigergrass, Miscanthus and bulrush species.

The sustained embryogenic cell culture utilized by the presented methodsis unlimitedly totipotent. During proliferation, each cell is a unit ofmultiplication and can undergo embryogenesis independently allowingcellular level selection for differential growth under selectiveconditions. In contrast, in cultures where somatic embryos or multipleshoot primordia are induced directly from an explant in a suspension orin a semi-solid secondary culture (e.g., U.S. Pat. No. 7,052,912(hereinafter “the '912 patent”)) the totipotent unit number is verylimited (i.e., the number of individuals) as compared to the totipotentembryogenic cell culture of the present invention where every cell is apotential individual. In the '912 patent, the multiplication, which isgenerated from secondary formation of embryos or adventitious shootmeristems comprising hundreds or thousands of cells, is limited to amuch lower rate because the individual plants regenerated are notoriginated at the cellular level from individual cells. This not onlyaffects the number of potential individual plants but also theefficiency of sorting out of novel or elite lines for breeding.

The embryogenic cell culture induction methods of the present inventionwere developed for a diverse group of monocot species, including Arundodonax. The method is based in part on the present inventors' discoveryof the embryogenic growth inducing effect of cold pre-treatment of shoottips containing immature inflorescences. While not wishing to be boundby any particular theory of the invention, it appears that the coldpretreatment not only induces physiological changes that cause theretention and increase of competence for induction of embryogenic cellcultures but also delays senescence and opens a wider window forprocessing the plant material. The invention is further based on theunexpected discovery that the transfer from media providing acombination of auxins and cytokinins to a media providing onlycytokinin, such as the synthetic cytokinin, thidiazuron, results in thepromotion of shoot elongation and root formation.

The sustained embryogenic cell culture of the present invention iscomposed of dividing cells which can produce unipolar embryos. In thepresence of light, the unipolar embryos germinate precociously and formmultiple shoot apical meristems and shoot primordia. On tertiary medium,the partial elongation of the shoots is accompanied by rooting. Thus,the methods of the present invention are suitable for not only forsustained maintenance and micropropagation of the totipotent cellculture lines but also for maintenance of sustained regenerating tissueculture lines as well.

Initiation and Production.

The present invention provides a method of producing and maintaining atotipotent embryogenic cell culture of a monocotyledonous plantcomprising: (a) cultivating an explant of tissue from a monocotyledonousplant shoot tip on a primary medium, wherein the explant has beenpretreated with a cold temperature and the primary medium comprisesauxin or auxin and cytokinin, to produce a totipotent embryogenic cellculture; (b) treating the totipotent embryonic cell culture with a coldtemperature; and (c) maintaining the totipotent embryogenic cell cultureby further cultivation on the primary medium and/or on a secondarymedium, whereby a totipotent embryogenic cell culture of amonocotyledonous plant is produced and maintained.

In some embodiments, the totipotent cell culture that is generated inthe primary cultivation step is maintained on the same medium and underthe same conditions in order to continue the generation of totipotenttissue. In other embodiments, the totipotent cell culture that isgenerated in the primary cultivation step is transferred to a secondarymedium for sustained proliferation of the totipotent cell culture.Examples of growth of A. donax on secondary culture medium are providedin FIG. 1.

In further embodiments, the totipotent cell culture is subjected to coldtreatment on secondary medium in order to increase growth rate. Inanother aspect of the invention, a method of micropropagating a monocotplant is provided wherein the totipotent cell culture is transferredfrom secondary medium to tertiary medium and allowed to form partiallyelongated shoots with roots (plantlets). The resulting plantlets can becultivated further on a quaternary medium free of hormones, vitamins andsugars for further growth and acclimatization to non-sterile andphotosynthetic conditions, thereby producing an acclimatized plantlet orplant. The plantlets can be transferred from the quaternary medium tosoil for further acclimatization. Alternatively, the plantlets can betransferred directly to soil from the tertiary medium in order toproduce an acclimatized plantlet or plant of the present invention.Following acclimatization, the plantlets or plants can be transplantedto any desired location. A representative embodiment of themicropropagation methods of the present invention is presented in FIG.2.

The shoot tip of the present invention includes, but is not limited to,an inflorescence, an immature inflorescence and an immatureinfructescence comprising immature to mature flowers and immaturefruits. The explant or shoot tip of living tissue from a monocot plantcan be obtained from any source, including, but not limited to, wildmonocot plants, cultivated monocot plants (greenhouse and field grown),and monocot plants regenerated from explants using the methods of thepresent invention. A monocotyledonous plant of the present inventionincludes, but is not limited to, Arundo spp., Thysanolaena spp.,Miscanthus spp., and Sciipus spp., and combinations thereof. Thus, amonocotyledonous plant of the present invention includes, but is notlimited to, giant reed (Arundo donax), tigergrass (Thysanolaena maxima),the silvergrasses (Miscanthus×giganteus and Miscanthus floridulus), andthe bulrushes (Scirpus californicus and Scirpus validus).

In the case of monocots of the grass family, in particular, giant reed(Arundo donax), tigergrass (Thysanolaena maxima), and the silvergrasses(Miscanthus×giganteus and Miscanthus floridulus), the explant can be animmature inflorescence. In one particular embodiment, the explant isobtained from the tips of preflowering shoots with leaf sheathscompletely enclosing the developing, but yet unemerged immatureinflorescence. In particular embodiments, an immature inflorescenceenclosed in leaf sheaths exhibits a higher yield of regenerable tissuethan other grass tissue sources.

An explant obtained from a living bulrush plant, in particular, Scirpuscalifornicus and Scirpus validus, is obtained, for example, from theshoots early in flowering (e.g., without open flowers or mostly withunopened flowers, open flowers and developing fruits; also referred toas an immature inflorescence or infructescence). In the case of bulrush,in particular embodiments, the immature inflorescence or infructescenceexhibits a higher yield of regenerable tissue than other bulrush tissuesources.

In an exemplary method of preparing the explant of a living monocotplant of the grass family for cultivation, in particular giant reed orthe silvergrasses, all but the terminal leaf sheaths are carefullystripped so as not to expose the inflorescence. In an exemplary methodof preparing the explant of a living monocot plant of the bulrush typefor cultivation, in particular Scirpus californicus and Scirpus validus,the branches of the inflorescence or the infructescence are cut off.Other methods of preparing explants will be apparent to those skilled inthe art.

The shoot tips of the grasses and the bulrushes can be sanitized, orsurface sterilized. One exemplary method of surface sterilization isimmersion of the shoot tips in a solution of 5 times diluted commercialbleach containing 10% v/v ethanol and a surfactant, for example, 0.1%Tween 80, for 15 minutes. The shoot tips are then rinsed three timeswith sterile water prior to further use. Such sterilization reduces oreliminates environmental microbial contamination. The inflorescence ofthe grass is then excised away from all leaf sheaths under asepticconditions and is cut or chopped into cross-sectional pieces. Followingsterilization, the inflorescence of the bulrush is cut or chopped intocross-sectional pieces under aseptic conditions. Any sterilized sharpblade, knife, or scalpel can be used for the cutting of theinflorescence. By cutting an aseptic immature inflorescence containing anumber of meristematic regions into cross-sectional pieces, theformation of regenerable tissue is induced.

In some embodiments, the explants are pretreated with a coldtemperature. Such a cold pretreatment of the explant can result in ahigher yield of regenerable tissue than those explants that are usedimmediately after excising or those that are stored at ambienttemperature. The duration of the cold pretreatment can be from about 1day to about 120 days. Thus, the cold treatment can be one day, oneweek, two weeks, three weeks, one month, two months, three months andfour months and the like. In further embodiments, the cold pretreatmentcan be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 95, 96, 97, 98, 99, 100, 105, 110, 115, or 120 daysand the like. Accordingly, the cold pretreatment can be from about oneday to about 14 days, one day to about 30 days, one day to about 45days, one day to about 60 days, one day to about 90 days, or one day toabout 105 days, and the like. In other embodiments, the coldpretreatment can be from about 7 days to about 14 days, about 7 days toabout 30 days, about 14 days to about 30 days, about 14 days to about 45days, about 14 days to about 60 days, about 30 days to about 45 days,about 30 days to about 60 days, about 30 days to about 90 days, about 45days to about 60 days, about 45 days to about 90 days, about 45 days toabout 120 days, about 60 days to about 90 days, 60 days to about 120days, or about 90 days to about 120 days, about 90 days to about 180days, and the like. In a particular embodiment, the cold pretreatment isa duration of 14 days (two weeks).

In representative embodiments, the pretreatment temperature can be in arange from about 0° C. to about 10° C. Thus, the pretreatmenttemperature can be about 0° C., 1° C., 2° C., 3° C., 4° C., 5° C., 6°C., 7° C., 8° C., 9° C., or 10° C. or any combination thereof. In otherembodiments, the pretreatment temperature can be in a range from 0° C.to about 5° C., from about 0° C. to about 8° C., from about 2° C. toabout 10° C., from about 2° C. to about 8° C., from about 2° C. to about5° C., from about 3° C. to about 5° C., from about 3° C. to about 7° C.,from about 3° C. to about 10° C., from about 5° C. to about 8° C., fromabout 5° C. to about 10° C., or from about 7° C. to about 10° C., fromabout 8° C. to about 10° C., and the like. In a further embodiment, thepretreatment temperature is 5° C. In some embodiments, the pretreatmentat a cold temperature is done in the presence of light. In a specificembodiment, the pretreatment is done in the presence of dim light. Dimlight as used herein is a light intensity of less than 30 micromoles m⁻²s⁻¹.

In particular embodiments, the pieces of the cut-up inflorescence orinfructescence are cultivated in a primary cultivation step in whichtotipotent tissue is generated. In some embodiments, the primarycultivation is carried out in the dark and, optimally, at approximatelyroom temperature. In other embodiments, the primary cultivation step iscarried out in the light and at approximately room temperature. Theinventors have found that the cultures exhibit increased multiplicationrates and increased longevity when maintained in the dark over thosecultures maintained in the light.

The primary cultivation step can be carried out for any period of timeand at any temperature sufficient to generate the totipotent embryogenictissue culture. In representative embodiments, the duration of theprimary cultivation step is from about one week to several months. Thus,the duration of the primary cultivation step can be about one week, twoweeks, three weeks, one month, two months, three months and four monthsand the like. In some embodiments the duration of the primarycultivation step is about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 95, 96, 97, 98, 99, 100, 105, 110, 115, or 120 daysand the like. In one embodiment, the duration of the primary cultivationis about four weeks. In still further embodiments the duration of theprimary cultivation step is about one week to about three weeks, aboutone week to about four weeks, about one week to about five weeks, aboutone week to about six weeks, about one week to about seven weeks, aboutone week to about eight weeks, about one week to about ten weeks, aboutone week to about twelve weeks, about one week to about fourteen weeksor about one week to about sixteen weeks, one week to about eighteenweeks, and the like.

The temperature for the primary cultivation step is not critical and canbe any temperature suitable for producing a totipotent embryogenic cellculture. Accordingly, the temperature for the primary cultivation stepcan be in a range from about 15° C. to about 35° C. Thus, thetemperature for the primary cultivation step is about 15° C., 16° C.,17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C.,26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C.,35° C., or any combination thereof. In some embodiments, the temperaturerange for the primary cultivation step is about 15° C. to about 20° C.,about 15° C. to about 25° C., about 15° C. to about 30° C., about 20° C.to about 25° C., about 20° C. to about 30° C., about 20° C. to about 35°C. or about 25° C. to about 30° C., and the like. In a furtherembodiment, the temperature range for the primary cultivation step isabout 26° C. to about 28° C. In a still further embodiment, thetemperature range for the primary cultivation step is about 25° C.

The present invention additionally provides a method of treating atotipotent embryogenic cell culture with a cold temperature, the methodcomprising incubating the totipotent embryogenic cell culture of step(a) of claim 1 and exposing the cell culture to at least one coldtreatment. In representative embodiments, the totipotent cell culturesof step (a) are incubated in individually sealed culture dishes. Culturedishes of the present invention include any type of dish, flask and/orbottle and the like in which tissue culture cells can be grown. Theseinclude, but are not limited to, Petri dishes, tissue culture flasks,Erlenmeyer flasks, microtiter plates and multiple-well cell cultureplates. In some embodiments, the culture dishes can be sealed usingsealants including, but not limited to, food service film, parafilm,pallet wrap and the like.

In some embodiments, the treating of the totipotent embryogenic cellculture with a cold temperature occurs in the dark. In otherembodiments, the cold treatment can occur in the light or withintermittent light. The intensity of light can be dim (less than 30micromoles m⁻² s⁻¹). As one of ordinary skill in the art wouldunderstand, even in the dark grown cultures, exposure to light ispossible when the cultures are subcultured or otherwise handled.

In other aspects of the present invention, treating with a coldtemperature comprises treatment at a temperature in a range from about4° C. to about 10° C., about 4° C. to about 8° C., about 4° C. to about6° C., about 5° C. to about 8° C., about 5° C. to about 10° C., about 6°C. to about 10° C., about 6° C. to about 8° C., about 7° C. to about 10°C., about 8° C. to about 10° C., and the like. Thus, the cold treatmentcomprises treatment at a temperature of about 4° C., 5° C., 6° C., 7°C., 8° C., 9° C., 10° C., or any combination thereof.

The duration of the cold treatment can be about 1 to about 300 days, ormore. Thus, the duration of the cold treatment can be about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300 days, 310 days, 320 days, 330 days, 340 days, 350 days, 360days, or any combination thereof. In one embodiment, the duration of thecold treatment can be about 5 days to about 90 days. In otherembodiments, duration of the cold treatment can be about 5 days to about15 days, about 5 days to about 30 days, about 5 days to about 45 days,about 5 days to about 105 days, about 5 days to about 120 days, about 5days to about 150 days, about 5 days to about 175 days, about 5 days toabout 200 days, about 5 days to about 225 days, about 5 days to about250 days, about 5 days to about 275 days, about 5 days to about 300days, about 15 days to about 30 days, about 15 days to about 45 days,about 15 days to about 90 days, about 15 days to about 120 days, about15 days to about 150 days, about 15 days to about 175 days, about 15days to about 200 days, about 15 days to about 250 days, about 15 daysto about 300 days, about 30 days to about 45 days, about 30 days toabout 90 days, about 30 days to about 120 days, about 30 days to about150 days, about 30 days to about 175 days, about 30 days to about 200days, about 30 days to about 225 days, about 30 days to about 250 days,about 30 days to about 275 days, about 30 days to about 300 days, about30 days to about 325 days, about 45 days to about 60 days, about 45 daysto about 90 days, about 45 days to about 120 days, about 45 days toabout 150 days, about 45 days to about 200 days, about 45 days to about250 days, about 45 days to about 300 days, about 45 days to about 320days, about 90 days to about 120 days, about 90 days to about 150 days,about 90 days to about 175 days, about 90 days to about 200 days, about90 days to about 220 days, about 90 days to about 250 days, about 90days to about 300 days, about 90 days to about 350 days, about 90 daysto about 350 days, about 120 days to about 180 days, about 120 days toabout 250 days, about 120 days to about 350 days, about 180 days toabout 250 days, or about 180 days to about 350 days, and the like.

In additional embodiments, the totipotent embryogenic cell culture istreated with a cold temperature for up to 180 days without subculture.In a further embodiment, the totipotent embryogenic cell culture istreated with a cold temperature for up to 180 days with at least onesubculture.

The cold treatments of the explants and the established cultures wereunexpectedly found to result in an increased growth rate of theembryogenic cell mass with a multiplication rate of 5× to 8× as comparedto the 2× to 3× observed without the cold treatment. This dramaticincrease of efficiency is of commercial significance since mostindustrial scale operations are limited by the multiplication rate. Theobserved positive effect of the cold treatments was a particularlysurprising finding since many of the plants of the present invention arecold sensitive species.

A medium that is useful for the primary cultivation step (i.e., theprimary medium) can be any basal medium used for plant tissue culture.Such media are well known to those of skill in the art. In oneembodiment, the primary medium is supplemented with at least one planthormone (i.e., plant growth regulator). Examples of suitable planthormones include auxins and cytokinins. Auxins of the present inventioninclude not only auxin but also any compound with auxin-like activity.Thus, auxins of the present invention include, but are not limited to,2,4-dichlorophenoxyacetic acid, picloram, and indolebutyric acid andcombinations thereof. Cytokinins of the present invention include notonly cytokinin but also any compound with cytokinin-like activity. Thus,cytokinins of the present invention include, but are not limited to,thidiazuron, zeatin, benzyladenine, kinetin, adenine hemisulfate anddimethylallyladenine, and combinations thereof. In some embodiments, theprimary medium is supplemented with at least one auxin and at least onecytokinin.

In representative embodiments, the primary medium also comprises acarbon source, which can be any carbon source appropriate for planttissue culture. Such carbon sources are known to those of ordinary skillin the art (Slater et al. Plant Biotechnology, the Genetic Manipulationof Plants, Oxford University Press, 368 pp., (2003)) and include, butare not limited to, sugars such as sucrose, glucose, maltose, galactoseand sorbitol, and the like. In the present invention, the preferredcarbon source is sucrose.

In one embodiment of the present invention, the primary medium isprepared by adding to sterile water: (a) MS (Murashige and Skoog, 1975)basal salts (Sigma Fine Chemicals, St. Louis, Mo.), 4.3 g/l; (b)Miller's salt solution (6% w/v, KH₂PO₄), 3 ml; (c) myo-inositol, 100mg/l; (d) Vitamix (Marton and Browse, Plant Cell Reports, 10: 235-239(1991), 2 ml.; (e) sucrose, 30 g/l; (f) supplemented with the plantgrowth regulators: (i) adenine hemisulfate, 80 mg/l; (ii) picloram, 0.12mg/l; (iii) indole-3-butyric acid, 1.0 mg/l; (iv)2,4-dichlorophenoxyacetic acid, 0.5 mg/l; (v) dimethylallyladenine, 0.5mg/l; (vi) zeatin, 0.5 mg/l; and (vii) thidiazuron, 3 mg/l.

In some embodiments, the medium is solidified with a gellant. Gellantsof the present invention include, but are not limited to, gellan gum,PHYTAGEL™, GELCARIN®, GELRITE®, food grade gellan gum, agarose, and thelike. The gellant used to solidify the primary medium can be used atconventional concentrations. In one embodiment, the medium is solidifiedwith PHYTAGEL™, 2 g/l.

In an additional embodiment of the present invention, the auxin in theprimary medium is 2,4-dichlorophenoxyacetic acid, which is present in aconcentration of about 0.2 mg/l, and the cytokinin in the primary mediumis thiadiazuron, which is present in a concentration of about 0.02 mg/l.

In further embodiments of the present invention, the pH of the mediumfor the primary cultivation step is adjusted to 5.6-5.8 before themedium is sterilized. In a further embodiment, the pH of the medium forthe primary cultivation step is adjusted to 5.8 before the medium issterilized. In one embodiment, the medium is sterilized in an autoclaveor pressure cooker for 25-35 minutes at a temperature of about 105° C.to about 121° C. In another embodiment, the temperature at which themedium is sterilized is about 109° C.

The warm medium may be poured into a sterile culture dish and allowed tocool to room temperature. The chopped explant material can bedistributed upon the surface of the gelled medium, and the culture dishcovered with a lid and sealed to preserve sterility. The culture dishcan be sealed with a strip of film such as food service film, parafilm,pallet wrap, and the like. The covered, sealed dish can then be placedin a location suitable for maintaining the temperature, as discussedabove.

In yet further embodiments of the present invention, the tissue beingcultured is kept in the dark during the primary cultivation step. Inother embodiments, the tissue can be maintained in the light during theprimary cultivation step. Exposure to light includes, but is not limitedto, continuous illumination and intermittent illumination. Illuminationcan include natural light (e.g., greenhouse), or artificial light. Theartificial light can be a mixture of incandescent light and cool whitefluorescent tubes. If continuous artificial illumination is employed,the intensity can be in a range from about 30 μmol m⁻² s⁻¹ to about 200μmol m⁻² s⁻¹ or more.

During the primary cultivation step totipotent embryogenic cell cultureforms from the cut-up explant tissue, which can be further subculturedand maintained on the primary or secondary culture medium. Certainsectors of the cell culture can give rise to unipolar embryos which formmultiple apical shoot meristems without significant shoot elongation.The culture at this point comprises both totipotent cell culture andtissue culture sectors (also referred to herein as totipotent orregenerable tissue culture). The totipotent tissue culture sectors canbe transferred to fresh primary cultivation medium for sustainedmaintenance or they may be transferred to secondary cultivation mediumfor sustained propagation. Alternatively, totipotent tissue culturesectors can be transferred to tertiary medium under light to inducegreening and rooting and further propagation or they can be transferredto hormone-free quaternary medium under light for the development ofroot systems and fully elongated shoots. Therefore, the totipotenttissue can be used as a regenerable source of genetic material forsustained maintenance and propagation. FIG. 3A shows Arundo donax in theprimary cultivation step.

Micropropagation, Scaling Up and Storage.

The totipotent embryogenic cell culture obtained in the primarycultivation step of the present invention can be maintained indefinitelyon the primary cultivation medium with regular passage to fresh medium.In other embodiments, the totipotent embryogenic cell culture obtainedin the primary cultivation step is transferred to a secondarycultivation medium in order to obtain a more friable embryogenic cellculture, which can also be maintained indefinitely on the secondarymedium with regular passage to fresh medium. FIG. 3B shows a totipotentembryogenic cell culture of A. donax on secondary cultivation medium.FIG. 3C shows the formation of unipolar nonzygotic embryos of A. donaxon secondary cultivation medium. Sustained totipotent embryogenic cellcultures of Miscanthus×giganteus on secondary culture media are shown inFIGS. 4A and 4B. FIG. 5 provides further examples of different monocotspecies produced using the methods described herein (Miscanthusfloridulus (FIG. 5A); Thysanolaena maxima (FIG. 5B); Scirpus validus(FIG. 5C); Scirpus californicus (FIG. 5D)).

In some embodiments, the totipotent embryogenic cell cultures aremaintained on the primary cultivation media or on secondary cultivationmedium in the dark. In other embodiments, the totipotent embryogeniccell cultures are maintained on the primary cultivation media or onsecondary cultivation medium in the light. When the cultures are grownin the presence of light, the light may be continuous or intermittent.Illumination can further include natural light (e.g., greenhouse) orartificial light. The artificial light can be a mixture of incandescentlight and cool white fluorescent tubes. The intensity of the light usedfor culturing the tissue on secondary media can be in a range from about30 μmol m⁻² s⁻¹ to about 200 μmol m⁻² s⁻¹ or more. Further, theintensity of light can be dim (less than 30 micromoles m⁻² s⁻¹). One ofordinary skill in the art would recognize that even in the case of thedark grown cultures, exposure to light is possible when the cultures aresubcultured or otherwise handled.

Accordingly, the present invention provides a method wherein aftercompletion of the primary cultivation step, the totipotent tissue iscultivated in a secondary cultivation step during which multiplicationcontinues. A medium that is useful for the secondary cultivation step(i.e., the secondary medium) can be any basal medium used for planttissue culture. Such media are well known to those of skill in the art.In one embodiment, the secondary medium is supplemented with at leastone plant hormone (e.g., plant growth regulator). Examples of suitableplant hormones include auxins and cytokinins.

Auxins of the present invention include not only auxin but also anycompound with auxin-like activity. Thus, auxins used with the secondarymedia include, but are not limited to, 2,4-dichlorophenoxyacetic acid,picloram and indolebutyric acid and combinations thereof. Cytokinins ofthe present invention include not only cytokinin but also any compoundwith cytokinin-like activity. Thus, cytokinins to be used with thesecondary media include, but are not limited to, thidiazuron, zeatin,benzyladenine, kinetin, adenine hemisulfate and dimethylallyladenine andcombinations thereof. In some embodiments, the secondary medium issupplemented with at least one auxin and at least one cytokinin.

The secondary medium also comprises a carbon source, which can be anyappropriate carbon source for tissue culture. Such carbon sources areknown to those of ordinary skill in the art (Slater et al. PlantBiotechnology, the Genetic Manipulation of Plants, Oxford UniversityPress, 368 pp., (2003)) and include, but are not limited to, sugars suchas sucrose, glucose, maltose, galactose and sorbitol, and the like. Insome embodiments of the present invention, the carbon source is sucrose.

In one embodiment of the present invention, the secondary medium isprepared by adding to sterile water: (a) MS (Murashige and Skoog, 1975)basal salts (Sigma Fine Chemicals, St. Louis, Mo.), 4.3 g/l; (b)Miller's salt solution (6% w/v, KH₂PO₄), 3 ml; (c) myo-inositol, 100mg/l; (d) Vitamix (Marton and Browse, Plant Cell Reports, 10: 235-239(1991), 2 ml.; (e) sucrose, 30 g/l; (f) supplemented with the plantgrowth regulators: (i) adenine hemisulfate, 400 μM; (ii)2,4-dichlorophenoxyacetic acid, 0.2 mg/l; (iii) thidiazuron, 0.01 μM. Insome embodiments, the medium is solidified with a gellant. Gellants ofthe present invention are described above. In one embodiment, thesecondary medium is solidified with PHYTAGEL™, 2 g/l.

In some embodiments of the present invention, the pH of the medium forthe secondary cultivation step is adjusted to 5.6-5.8 before the mediumis sterilized. In a further embodiment, the pH of the medium for thesecondary cultivation step is adjusted to 5.8 before the medium issterilized. The medium is sterilized in an autoclave or pressure cookerfor 25-35 minutes at a temperature of about 105° C. to about 121° C. Insome embodiments, the temperature at which the medium is sterilized isabout 109° C.

The temperature for the secondary cultivation step is not critical andcan be any temperature suitable for sustaining the proliferation of thetotipotent cell culture. In representative embodiments, the temperaturefor the secondary cultivation step is in a range from about 15° C. toabout 35° C. Thus, the temperature for the secondary cultivation step isabout 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C.,23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C.,32° C., 33° C., 34° C., 35° C., or any combination thereof. In someembodiments, the temperature range for the secondary cultivation step isabout 15° C. to about 20° C., about 15° C. to about 25° C., about 15° C.to about 30° C., about 20° C. to about 25° C., about 20° C. to about 30°C., about 20° C. to about 35° C. or about 25° C. to about 30° C., andthe like. In a further embodiment, the temperature range for thesecondary cultivation step is about 26° C. to about 28° C. In a stillfurther embodiment, the temperature range for the secondary cultivationstep is about 25° C. FIGS. 1A-C and FIGS. 3B-C show Arundo donax in thesecondary cultivation stage. Miscanthus×giganteus in the secondarycultivation stage is pictured in FIGS. 4 a and 4B, while Miscanthusfloridulus in the secondary cultivation stage is shown in FIG. 5A. FIG.5B illustrates Thysanolaena maxima in the secondary cultivation stage.

The totipotent cell culture can be maintained and propagatedindefinitely on secondary medium. Similar to the totipotent cell culturemaintained on primary medium, the multiplication rate of the totipotentcell culture maintained on secondary medium is increased by treatmentwith a cold temperature. Thus, in particular embodiments, methods forcold treatment of the totipotent cell culture on secondary medium arethe same as provided above for the totipotent cell culture on primarymedium. According to particular embodiments of the present invention,the secondary medium is preferred for the maintenance and the coldtreatment of the totipotent cell culture.

In further embodiments of the present invention, the cold treatments areincorporated into the micropropagation cycle. Accordingly, in oneembodiment, a micropropagation cycle for the embryogenic cell culture isprovided wherein the micropropagation cycle (subculture intervals) is2-6 weeks in the dark at ambient temperature with intercalation of acold treatment in the dark at 4-10° C. In representative embodiments,the cold treatment comprises treating the embryogenic cell culture witha cold temperature for a period in a range of about one month to aboutten months, or longer. Thus, the cold treatment can be about one, two,three, four, five, six, seven, eight, nine, ten, eleven or twelvemonths, and the like. In particular embodiments, the cold treatment canbe about one week, two weeks three weeks, four weeks, five weeks, sixweeks, seven weeks, or eight weeks, and the like. In furtherembodiments, the cold treatment can be about 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 145, 150, 160, 170, 180, 190, 200, 205, 210,215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280,285, 290, 295, 300, 305, 310, 315, 320, 330, 335, 340, 345, 350, 355,360, 365 days, or any combination thereof. In further embodiments, thecold treatment can be in a range from about 2 weeks to about 6 weeks,about 2 weeks to about 8 weeks, about 2 weeks to about 10 weeks, about 2weeks to about 12 weeks, about 2 weeks to about 14 weeks, about 2 weeksto about 16 weeks, about 4 weeks to about 8 weeks, about 4 weeks toabout 12 weeks, about 4 weeks to about 16 weeks, about 4 weeks to about20 weeks, about 4 weeks to about 24 weeks, about two months to aboutfour months, about two months to about six months, about two months toabout eight months, about two months to about ten months, about twomonths to about twelve months, about four months to about six months,about four months to about eight months, about four months to about tenmonths, about four months to about twelve months, about six months toabout eight months, about six months to about ten months, about sixmonths to about twelve months, or about six months to about fourteenmonths, and the like. In a particular embodiment, the cold treatment isin a range from about 4 weeks to about 6 weeks. The micropropagationcycle is useful with any of the media on which the totipotent cellculture is maintained and propagated.

The present invention further provides a method of prolonged orlong-term cold treatment (i.e., cold storage) of the embryogenic cellcultures established on the secondary medium. Thus, the presentinvention further provides a method wherein the totipotent embryogeniccell culture can be stored long-term at a cold temperature. Accordingly,cultures produced in excess of the minimum mass required for maintenancecan be maintained and stored at a cold temperature in order to build upstock. In some embodiments, the cold storage occurs in the dark. Inother embodiments, the cell cultures are stored at a cold temperature inthe light. In particular embodiments, the cultures can be maintained onspent medium. As previously described, the inventors have found that thecultures maintained in the dark while in cold storage exhibit increasedrates of multiplication rates and increased longevity over thosemaintained in the light. The duration and temperature of the long-termor prolonged cold storage is the same as that described above for thecold treatments of the totipotent embryogenic cell cultures.

In additional embodiments, the totipotent embryogenic cell culture,which is being stored long-term at a cold temperature, is subculturedduring the storage. In other embodiments, the totipotent embryogeniccell culture, which is being stored long-term at a cold temperature isnot subcultured during the storage. Cultures that are maintained instorage for a prolonged time or long-term need to be “rehabilitated.”Rehabilitation refers to transferring of the stored cultures to freshmedia. In some embodiments, when the cultures are brought out ofstorage, the subculturing or transfer to fresh media occurs every two tosix weeks. In other embodiments, the subculturing can occur about everyfour to six weeks, about every one to eight weeks or about every two toeight weeks, and the like. In further embodiments, the subculturingoccurs about every week, about every two weeks, about every three weeks,about every four weeks, about every five weeks, about every six weeks,about every eight weeks, and the like. Once the cultures are brought outof storage, the subculturing is generally done on a regular basis.

Micropropagation as Multishoot Cultures.

After completion of the secondary cultivation step on the secondarymedium, the totipotent tissue can then be transferred to a tertiarymedium in which multiplication continues and complete plantlets areinduced. Thus, according to embodiments of the present invention amethod is provided wherein the totipotent embryonic cell culture istransferred from the secondary medium to a tertiary medium in order tocontinue multiplication of the cell culture and to produce a plantletwith roots and shoots. FIG. 3D shows plants of A. donax regenerated fromthe embryogenic cell culture and multiplying in tertiary culture medium.Examples of Miscanthus×giganteus plants regenerated from the embryogeniccell culture on tertiary medium are provided in FIGS. 4C, 4D and 4E.FIG. 5D shows Scirpus californicus plants regenerated from theembryogenic cell culture on tertiary medium.

A medium that is useful for the tertiary cultivation step (i.e.,tertiary medium) can be any basal medium used for plant tissue culture.Such media are well known to those of skill in the art. In oneembodiment, the tertiary medium is supplemented with at least one planthormone (e.g., plant growth regulator). Examples of plant hormonessuitable for the tertiary medium include cytokinins and compounds withcytokinin-like activity. Thus, in some embodiments, the only hormonepresent in the tertiary medium is cytokinin. Cytokinins useful in thepresent invention include, but are not limited to, thidiazuron, zeatin,benzyladenine, kinetin, adenine hemisulfate and dimethylallyladenine,and combinations thereof. In one embodiment, the tertiary medium issupplemented with thidiazuron. The present invention additionallyprovides a method wherein the plant hormone is supplemented in thetertiary medium at a concentration that is lower than that used in theprimary medium.

The inventors note that the use of the synthetic cytokinin, thidiazuron,in the present invention resulted in a surprising effect. As previouslydiscussed, embryogenic callus is typically obtained from monocots byinducing the primary cell culture on a medium containing one or moreauxin or auxin-type plant hormones followed by a secondary cultivationstep on a lowered auxin concentration but in the presence of a cytokininor cytokinin-type plant hormone. The embryogenic potential of monocotcell cultures produced by these methods diminishes over time and makingit necessary to repeatedly reinitiate the primary cell culture (U.S.Pat. No. 6,153,812 issued Nov. 28, 2000; Trigiano and Gray, Plant tissueculture concepts and laboratory exercises. Second ed., Boca Raton: CRCPress (2000)). In contrast, the hormone combination taught in thepresent invention has an unexpected result in that the primary callusinduced from the explant already shows embryogenesis without the needfor a secondary cultivation step and that the embryogenic capacity doesnot diminish over time.

As known to those of skill in the art, cytokinins, both natural andsynthetic, are used to induce shoot formation while inhibiting rootformation (Trigiano and Gray, Plant tissue culture concepts andlaboratory exercises. Second ed., Boca Raton: CRC Press (2000)).However, in the present invention, the use of cytokinin, such as thesynthetic cytokinin thidiazuron, produced a surprising effect in thatupon transfer from the primary cultivation medium containing acombination of auxins and cytokinins to a medium without auxin butmaintaining the cytokinin (e.g., thidiazuron) not only was shootelongation promoted but unexpectedly root formation also resulted.

In some embodiments, the medium for tertiary cultivation (i.e., thetertiary medium) as provided by the present invention comprises a carbonsource. The carbon source for the tertiary medium can be any appropriatecarbon source known to those of ordinary skill in the art (Slater et al.Plant Biotechnology, the Genetic Manipulation of Plants, OxfordUniversity Press, 368 pp., (2003)). Carbon sources useful for thetertiary medium include, but are not limited to, sugars such as sucrose,glucose, maltose, galactose and sorbitol, and the like. Similarly to theprimary and secondary media, the preferred carbon source for thetertiary medium is sucrose.

In one embodiment, the tertiary medium is prepared by adding to sterilewater (a) the cytokinin, thidiazuron; (b) sucrose, 30 g/l; (c) Miller'ssalt solution (6% w/v, KH₂PO₄), about 3 ml; and (d) MS salts, 4.3 g/l.In one embodiment the concentration of the thidiazuron is from about0.01 mg/l to about 1 mg/l. In another embodiment, the concentration ofthe thidiazuron is about 0.02 mg/l.

The tertiary medium can be gelled and sterilized as described for theprimary and secondary medium. Totipotent tissue from the secondarycultivation step can then be used to inoculate the tertiary medium. Theinoculated tertiary cultivation medium is then cultured, under any lightregime in order to obtain rooted plants. The tertiary cultivation stepcan be carried out for any period of time or at any temperaturesufficient to obtain elongation of shoots and rooted plants. In oneembodiment, the culture conditions include continuous light, about roomtemperature, for a period of from about one week to about four weeks.

In further embodiments, the tertiary medium comprises the cytokininthidiazuron. In still further embodiments, the thiadiazuron is presentin the tertiary medium in a concentration of about 0.02 mg/l.

The temperature and the duration of the tertiary cultivation step is notcritical and can be any temperature or duration sufficient for theproduction of complete plantlets with roots and partially elongatedshoots. In representative embodiments, the temperature and duration forthe tertiary cultivation step is similar to that described above for theprimary cultivation step.

In the case of the monocot plants of the present invention (for example,Arundo donax, Thysanolaena maxima, Miscanthus×giganteus, Miscanthusfloridulus, Scitpus validus and Scitpus californicus), the tertiarycultivation step results in complete plantlets with roots and partiallyelongated shoots that conveniently fit the commonly used plantpropagation containers. Further, the resulting tertiary cultures do notinterfere with handling of the plantlets with the tools used fortransfer during division of the cultures.

The embryogenic cell culture obtained in the primary cultivation stepcan be maintained indefinitely on the tertiary cultivation medium withregular passage to fresh media. The method is therefore suitable notonly for sustained maintenance and propagation of the totipotent cellculture lines but for maintenance of sustained regenerating tissueculture lines as well.

Acclimatization and Planting.

Upon completion of the tertiary cultivation step, the plantlets can bemoved either directly to soil for acclimatization, or they can betransferred to a quaternary medium to permit gradual acclimatization tonon-sterile and photoautotrophic conditions. Acclimatization generallyinvolves an introduction of a plantlet to ambient air conditionsincluding moisture, temperature and non-sterility. In one embodiment,acclimatization comprises gradually removing the lids of the culturedishes to expose the plantlets on quaternary media to lower humidity andnon-sterile conditions. Acclimatization typically further comprisesstimulating the plantlet to begin photosynthesizing by the fact that thequaternary media and/or the soil do not provide a freely availablecarbon source such as sucrose.

In one embodiment, the plantlets are transferred from the tertiarymedium to a quaternary medium that is similar to the medium used for thetertiary cultivation step, but which is free of plant hormones, vitaminsand a carbon source.

The quaternary cultivation step can be carried out for any period oftime and at any temperature sufficient to acclimatize the plantlets.Thus, in representative embodiments, the temperature for the quaternarycultivation step is similar to that described for the primary andsecondary cultivation steps. In particular embodiments, the quaternarycultivation step can be carried out at substantially room temperature.

In an exemplary embodiment, the duration of the quaternary step can bein the range from about one week to about four weeks or more. In furtherembodiments, the duration of the quaternary step can be one, two, three,and four weeks, and the like. In additional embodiments, the durationcan be one month, two months, three months or more. In still furtherembodiments, the duration for the quaternary step is from about one weekto about three weeks, about one week to about five weeks, about one weekto about six weeks, about one week to about eight weeks, about two weeksto about four weeks, about two weeks to about six weeks, about two weeksto about eight weeks, about two weeks to about twelve weeks, or aboutfour weeks to about eight weeks, and the like.

A medium that is useful for the quaternary cultivation step (i.e., thequaternary medium) can be any basal medium used for plant tissueculture. Such media are well-known to those of skill in the art andinclude, but are not limited to, MS medium, and Gamborg's B5 medium(full or ½ strength) without a carbon source, vitamins, hormones or anyother additive, such as nutrient mixtures, buffers, antifungalcompounds, amino acids, organic acids, nucleotides, nucleosides, bases,hardening agents and the like.

An example of A. donax plants transferred to pots from quaternary mediumfollowing acclimatization is provided in FIG. 3E. FIG. 5C shows Scirpusvalidus in the acclimatization stage.

Transportation.

Biotechnological field applications such as habitat restoration,remediation and biomass production require the production of propagulesat an industrial scale. Depending on the scale, part or all of thepropagation technology needs to be deployed to a location convenient tothe vicinity of the application. Thus, storage and mobilization ofstockpiles of propagules in the appropriate developmental stage arenecessary for these applications.

For industrial-scale planting, the present invention provides methodswherein the cultures, plantlets or plants from any of the variouscultivation steps described herein can be moved near the location offinal planting. In some embodiments of the present invention, aftercompleting the secondary cultivation step, the culture dishes that arefilled with the totipotent tissue are accumulated in cold-storage in asufficient number for a particular planting project. The dishes can betransported by any means of transportation in a cold compartment to asuitable propagation facility where they can then be unpacked andpropagated further as needed or transferred to tertiary medium.

The compartment in which the cultures, plantlets and plants aretransported can be insulated or refrigerated. When the transportationtime is less than 3 weeks, the compartment used to transport thecultures, plantlets or plants may be insulated and maintained at ambienttemperatures. When the time needed for transportation is three weeks ormore, or conditions otherwise require it, the cultures, plantlets orplants may be transported in compartments maintained at a coldtemperature. The temperatures of the cold compartment can be about thesame as those described above for the cold treatment and cold storage ofembryogenic cell cultures.

The plant materials of the present invention that can be transportedinclude any of the plant materials described herein including theembryogenic cell culture, tissue culture, plantlets, plants orpropagules. Thus, in one embodiment of the present invention, propagulesare transported as embryogenic cell cultures in culture dishes onsemisolid medium in the dark at ambient temperatures or at cold storagetemperatures. The types of propagules that can be transported include,but are not limited to, tertiary microtillering shoot cultures inliquid, tertiary microtillering shoot cultures on semisolid culturemedium in vessels or bags, microtillering multishoot cultures insugar-free liquid cultures in vessels, and acclimatized bare-root plantspackaged. The choice of propagule transported depends on the need,demand and conditions at the location.

The distances over which the plant materials can be transported can belong or short distances. Thus, the plant materials can be maintained intransit for about one day or less or for about 30 days or more.

Thus, as described herein, the cold treatments of the present inventionserve multiple purposes. Cold treatments can improve the longevity ofthe tissue culture, plantlets, plants and propagules, allow safe andcost effective transportation and/or accelerate totipotent growth oncethe plant materials are unpackaged and further cultured at the finaldestination site. FIG. 3F shows a stand of A. donax plants establishedfrom in vitro cloned plants.

Enhanced Somaclonal Breeding and Genetic Manipulation.

A major limiting factor associated with emerging biotechnologicalapplications of nontraditional crop plants is the lack of elite plantmaterial. Elite plant material is plant material selected for desirablegenotypic and phenotypic characteristics. As such, elite plant materialincludes, but is not limited to, vegetative clones, F1-hybrids, andinbred plant materials selected for desirable genotypic and phenotypiccharacteristics. Classical breeding technology predominantly relies onthe use of inbred (pure) lines, crosses, and analysis of progenies. Purelines are rarely available for environmentally important wild plantspecies and obtaining them is very tedious process. The use oftotipotent tissue cultures of the present invention to induce somaclonal(genetic and/or epigenetic) variation in clonal plant materials canprovide shortcuts and improved efficiency.

Accordingly, one embodiment of the present invention provides a methodof producing an elite plant line comprising: selecting for at least onetrait of interest in the totipotent embryogenic cell culture and/orplantlet produced by the methods of the present invention, wherein theat least one trait of interest is a result of somaclonal variation orthe introduction of at least one heterologous nucleotide sequence genomeof a cell of the totipotent embryogenic cell culture and/or plantlet,and cultivating the totipotent embryogenic cell culture and/or plantletcomprising the at least one trait of interest to produce an elite plantline.

The present invention provides further embodiments, wherein the at leastone trait of interest is resistance and/or increased tolerance to anenvironmental condition. In some embodiments, the increased tolerance isto the presence of a chemical pollutant in the environment. In otherembodiments, the resistance or increased tolerance is to a halogenatedphenol or other xenobiotic. In additional embodiments, the resistance orincreased tolerance is to salts.

In still further embodiments, other characteristics for which elitelines may be selected for include, but are not limited to, improvedmorphology, improved biomass parameters, improved pest and pathogentolerance, and any combination thereof. The improved biomass parametersinclude, but not limited to, cellulose/lignin ratio and fiber quality,and any combination thereof.

By increasing the length and volume of the totipotent phase, especiallythe embryogenic cell culture phase, somaclonal variations will beincreased; thus, allowing for a very efficient cellular level selectionof elite lines originated from somaclonal or gene transfer events. Insome embodiments, the totipotent sustained secondary cultures of thepresent invention further increase the efficiency of selection byextending the selection for traits, which are expressed only at thelevel of differentiated shoots. Furthermore, in some embodiments, analtered elite population can be obtained without generation andcharacterization of individual lines, if the selection pressure isconstantly applied to the embryogenic cultures until the plants from thesecondary cultures differentiate. The resultant altered elite populationcan be used directly in applications without further reducing thenatural genetic variation in the elite population by a second cloningevent.

The sustained totipotent cell cultures also provide an excellent systemfor all forms of molecular breeding including the introduction of genesof interest and complex genetic constructs, using genetic transformationfor genetic and epigenetic manipulation of these plants.

Thus, the present invention provides embodiments wherein the explant isselected from a transgenic plant stably transformed with at least oneheterologous nucleotide sequence. In other embodiments, at least oneheterologous nucleotide sequence is introduced into a cell of thetotipotent embryogenic cell culture grown on either the primary orsecondary medium to produce a stable transgenic totipotent embryogeniccell culture. Further embodiments comprise producing a transgenicplantlet and/or plant from the transgenic totipotent embryogenic cellculture.

Additional embodiments of the present invention comprise introducing atleast one heterologous nucleotide sequence of interest into a cell ofthe embryogenic cell culture grown on tertiary medium to produce astable transgenic embryogenic cell culture and/or a stable transgenicplantlet with shoots and roots. A further embodiment provides a methodof introducing at least one heterologous nucleotide sequence of interestinto a cell of a plantlet produced by the methods of the presentinvention grown on tertiary medium or quaternary medium to produce astable transgenic plantlet with shoots and roots and/or a stabletransgenic plant. In a still further embodiment, a method is providedwherein at least one heterologous nucleotide sequence is introduced intoa cell of the acclimatized plantlet or plant produced by the methods ofthe present invention in order to produce a stable transgenic plantletand/or plant.

The heterologous nucleotide sequence of interest can be any heterologousnucleotide sequence and includes, but is not limited to, nucleotidesequences that encode antibiotic resistance genes, pesticide resistancegenes, salt resistance genes, mercuric reductase genes, organomercuriallyase gene, Bt-toxin genes, and any combination thereof. The nucleotidesequences of interest of the present invention can also encode siRNAsthat down-regulate genes involved in lignin and polysaccharidebiosynthesis. Lignin and polysaccharide biosynthesis genes are known tothose of skill in the art (See, for example, Boerjan et al., Annu. Rev.Plant Biol. 54: 519-546 (2003) and Burton et al., Planta 221: 309-312(2005)). Thus, genes involved in lignin biosynthesis include, but arenot limited to, cinnamyl alcohol dehydrogenase, cinnamoyl coenzyme-Areductase, caffeic acid O-methyltransferase, caffeoyl-CoA O-methyltransferase, 4-coumarate-coenzyme A ligase, and the like. Genes involvedin polysaccharide biosynthesis include, but are not limited to,glycosyltransferases, cellulose synthase complex, endoglucanases, andthe like.

A nucleotide sequence can be introduced into a cell of an embryogeniccell culture or a plant cell by any method known to those of skill inthe art. Procedures for transforming a wide variety of plant species arewell known and routine in the art and are described throughout theliterature. Such methods include, but are not limited to, transformationvia bacterial-mediated DNA delivery, viral-mediated DNA delivery,silicon carbide or DNA whisker-mediated DNA delivery, liposome mediatedDNA delivery, microinjection, microparticle bombardment,electroporation, sonication, infiltration, PEG-mediated DNA uptake, anyother electrical, chemical, physical or biological mechanism thatresults in the introduction of DNA into the plant cell, and anycombination thereof. General guides to various plant transformationmethods known in the art include Miki et al. (“Procedures forIntroducing Foreign DNA into Plants” in Methods in Plant MolecularBiology and Biotechnology, Glick, B. R. and Thompson, J. E., Eds. (CRCPress, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska(Cell. Mol. Biol. Lett. 7:849-858 (2002)).

Bacterial mediated DNA delivery includes but is not limited to DNAdelivery by Agrobacterium spp. and is described, for example, by Mártonet al. (Nature 277:129-131 (1979)); Horsch et al. (Science 227:1229(1985); Ishida et al. (Nature Biotechnol. 14:745 750 (1996)); and Fraleyet al. (Proc. Natl. Acad. Sci. 80: 4803 (1983)). Transformation byvarious other bacterial species is described, for example, byBroothaerts et al. (Nature 433:629-633 (2005)).

Physical delivery of nucleotide sequences via microparticle bombardmentis described, for example, in Sanford et al. (Methods in Enzymology217:483-509 (1993)) and McCabe et al. (Plant Cell Tiss. Org. Cult.33:227-236 (1993)).

Another method for physical delivery of DNA to plants is sonication oftarget cells. This method is described, for example, in Zhang et al.(Bio/Technology 9:996 (1991)). Alternatively, liposome or spheroplastfusion can be used to introduce nucleotide sequences into plants.Examples of the use of liposome or spheroplast fusion are provided inDeshayes et al. (EMBO J., 4:2731 (1985), and Christou et al. (Proc Natl.Acad. Sci. U.S.A. 84:3962 (1987)). Direct uptake of DNA into protoplastsusing CaCl₂ precipitation, polyvinyl alcohol or poly-L-ornithine isdescribed, for example, in Hain et al. (Mol. Gen. Genet. 199:161 (1985))and Draper et al. (Plant Cell Physiol. 23:451 (1982)). Electroporationof protoplasts and whole cells and tissues are described, for example,in Donn et al. (In Abstracts of VIIth International Congress on PlantCell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin et al.(Plant Cell 4:1495-1505 (1992)); Spencer et al. (Plant Mol. Biol.24:51-61 (1994)) and Fromm et al. (Proc. Natl. Acad. Sci. 82: 5824(1985)). Polyethylene glycol (PEG) precipitation is described, forexample, in Paszkowski et al. (EMBO J. 3:2717 2722 (1984)).Microinjection of plant cell protoplasts or embryogenic callus isdescribed, for example, in Crossway (Mol. Gen. Genetics 202:179-185(1985)). Silicon carbide whisker methodology is described, for example,in Dunwell et al. (Methods Mol. Biol. 111:375-382 (1999)); Frame et al.(Plant J. 6:941-948 (1994)); and Kaeppler et al. (Plant Cell Rep.9:415-418 (1990)).

In addition to these various methods of introducing nucleotide sequencesinto plant cells, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable for carrying out the methods of this invention. See, forexample, Gruber et al., (“Vectors for Plant Transformation” in Methodsin Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson,J. E., Eds. (CRC Press, Inc., Boca Raton, (1993), pages 89-119)

Accordingly, in one embodiment, a heterologous nucleotide sequence isintroduced into a cell of the totipotent embryogenic cell culture orplant cell produced by the methods of the present invention byco-cultivation of the cell of the totipotent embryogenic cell culturewith Agrobacterium tumefaciens to produce a stable transgenic totipotentembryogenic cell culture or stable transgenic plant cell.

In a further embodiment, a method is provided, wherein a heterologousnucleotide sequence is introduced into a cell of the totipotentembryogenic cell culture or plant cell produced by the methods of thepresent invention by direct DNA transfer to produce a stable transgenictotipotent embryogenic cell culture or transgenic plant cell.

The methods of the present invention further provide producing a stabletransgenic plantlet and/or plant from the transgenic totipotentembryogenic cell culture or transgenic plant cell. Methods of selectingfor stably transformed transgenic cell culture, plant cells or plantsare routine in the art.

Production of Purpose-Oriented Plant-Microbe Associations.

The sustained aseptic totipotent cell cultures and multishoot culturesof the present invention also provide an excellent system for theproduction of microbial-plant associations before acclimatization andtransfer to soil. The microbial species or consortium is selected with aparticular application in mind and can be tuned for providing fitness ina particular planting location or for contributing facultative metabolicfunctions that, in synergy with the plant's own metabolism, provideenhanced metabolic capabilities that are useful for various applicationssuch as phytoremediation or raw material conversion.

Accordingly, the present invention provides a method of establishing aplant-microbe association comprising co-cultivating at least oneplantlet and/or plant produced by the methods of the present inventionwith at least one microbial species in a quaternary medium to establisha plant-microbe association. As described previously, the quaternarymedium of the present invention lacks plant hormones, a carbon sourceand vitamins. In one embodiment of the invention, one microbial speciesis co-cultivated with at least one plantlet and/or plant produced by themethods of the present invention to establish a plant-microbeassociation. In other embodiments, two or more microbial species areco-cultivated with at least one plantlet and/or plant produced by themethods of the present invention.

In representative embodiments, the at least one plant and/or plantlet isselected from plantlets growing on the tertiary medium. In otherembodiments, the at least one plantlet and/or plant is a plantlet orplant growing on quaternary medium. In still other embodiments, the atleast one plantlet or plant is a plant or plantlet that is growing onsoil. Additionally, the at least one plantlet or plant can be from eliteplant lines produced by the methods of the present invention.

Microbial species that can be used to establish a plant-microbeassociation include, but are not limited to, Pseudomonas mallei,Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas cepaica,Acinetobacter lwoffi, Acinetobacter baumanni, Bacillus licheniformis,Bacillus cereus and Phyllobacterium spp., and combinations thereof. Infurther embodiments of the invention, a plantlet having an establishedplant-microbe association is acclimated to non-sterile andphotosynthetic conditions.

Thus, in some embodiments of the invention the plant-microbialassociation is between Arundo donax and a microbe selected from thegroup consisting of Pseudomonas mallei, Acinetobacter baumanni,Acinetobacter lwoffi, Pseudomonas cepaica, Pseudomonas aeruginosa,Pseudomonas fluorescens, Bacillus cereus, Bacillus licheniformis, andPhyllobacterium spp., and combinations thereof.

In other embodiments, the plant-microbial association is betweenThysanolaena maxima and a microbe selected from the group consisting ofPseudomonas mallei, Acinetobacter baumanni, Acinetobacter lwoffi,Pseudomonas cepaica, Pseudomonas aeruginosa, Pseudomonas fluorescens,Bacillus cereus, Bacillus licheniformis, and Phyllobacterium spp., andcombinations thereof.

In further embodiments, the plant-microbial association is betweenMiscanthus×giganteus and a microbe selected from the group consisting ofPseudomonas mallei, Acinetobacter baumanni, Acinetobacter lwoffi,Pseudomonas cepaica, Pseudomonas aeruginosa, Pseudomonas fluorescens,Bacillus cereus, Bacillus lichenifonnis, and Phyllobacterium spp., andcombinations thereof.

In yet further embodiments, the plant-microbial association is betweenMiscanthus floridulus and a microbe selected from the group consistingof Pseudomonas mallei, Acinetobacter baumanni, Acinetobacter lwoffi,Pseudomonas cepaica, Pseudomonas aeruginosa, Pseudomonas fluorescens,Bacillus cereus, Bacillus lichenifonnis, and Phyllobacterium spp., andcombinations thereof.

In some embodiments of the present invention, the plant-microbialassociation is between Scirpus californicus and a microbe selected fromthe group consisting of Pseudomonas mallei, Acinetobacter baumanni,Acinetobacter lwoffi, Thysanolaena maxima, Pseudomonas cepaica,Pseudomonas aeruginosa, Pseudomonas fluorescens, Bacillus cereus,Bacillus licheniformis, and Phyllobacterium spp., and combinationsthereof.

In other embodiments, the plant-microbial association is between Scirpusvalidus and a microbe selected from the group consisting of Pseudomonasmallei, Acinetobacter baumanni, Acinetobacter lwoffi, Pseudomonascepaica, Pseudomonas aeruginosa, Pseudomonas fluorescens, Bacilluscereus, Bacillus licheniformis, and Phyllobacterium spp., andcombinations thereof.

After completing the co-cultivation step, the dishes can be transportedby any means of transportation to a suitable propagation facility wherethe plants can then be unpacked and acclimatized, and transplanted intosoil.

Phytoremediation.

The present invention further provides methods directed tophytoremediation. Phytoremediation can be used to remove pollutants fromthe environment including, but not limited to, xenobiotic compounds andother compounds or pollutants such as those present in the environmentin concentrations that are higher than normal.

Thus, in one embodiment, phytoremediation comprises establishing aplurality of plants produced by the methods of the present invention andpossessing the same genetic characteristics in a liquid medium, andcontacting the roots of the plants with an environmental pollutant inthe liquid medium, thereby causing the environmental pollutant to beremoved from the liquid medium.

In other embodiments, phytoremediation comprises establishing aplurality of plants produced by the methods of the present invention andpossessing the same genetic characteristics in a land area andcontacting the roots of the plants with an environmental pollutant inthe land area, thereby causing the environmental pollutant to be removedfrom the land area.

In representative embodiments, a plurality of plants comprises at leasttwo plants. For example, in some embodiments, a plurality of plantscomprises 10 plants. In further exemplary embodiments, a plurality ofplants comprises hundreds or thousands of plants.

The plants of the present invention that can be used in phytoremediationinclude but are not limited to those plants that are produced from wildplants, cultivated plants (greenhouse and field grown), and plantsregenerated from explants produced by the methods of the presentinvention. In addition, plants of the present invention that can be usedin phytoremediation comprise elite lines produced using the methodsdescribed herein.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES Example 1 Increased Multiplication Rate by Cold Treatment ofArundo Embryogenic Cell Culture

The multiplication rate of Arundo embryogenic cell culture on secondaryculture medium is increased by a six-month pretreatment in the cold aswell as by reducing the size of initial inoculum. An increase in theproportion of embryogenic cells is also apparent after cold treatmentresulting in about a 50% increase in yield. In the case of large-scalepropagation where subculturing is the rate limiting step (manual workunder sterile hood conditions), the cold treatments result in a dramaticincrease in efficiency. Table 1 shows the data for two levels of initialmass to medium ratios and further shows that a smaller initiation massresults in much higher multiplication rates.

TABLE 1 Initial mass to medium ratio of Arundo embryogenic cell cultureon secondary culture medium. Without cold treatment With cold treatmentIntial mass Final mass Fold Intial mass Final mass Fold [g] [g] Increase[g] [g] Increase 1.60 5.96 3.7 1.14 6.32 5.5 1.95 6.04 3.1 1.53 6.31 4.12.33 7.04 3.0 0.91 4.60 5.1 1.93 6.11 3.2 1.27 5.31 4.2 1.10 4.27 3.91.25 5.90 4.7 1.60 5.68 3.6 1.59 6.62 4.2 1.48 3.76 2.5 1.06 7.60 7.21.20 3.10 2.6 1.27 5.41 4.3 1.65 3.2 ± 0.2 1.25 4.9 ± 0.4 0.60 4.22 7.00.61 5.86 9.6 0.69 4.25 6.2 0.70 6.50 9.3 0.72 5.01 7.0 0.59 5.77 9.80.67 4.13 6,2 0.66 5.41 8.2 0.72 4.34 6.0 0.55 5.45 9.9 0.73 4.85 6.60.53 4.99 9.4 0.69 6.5 ± 0.2 0.61 9.4 ± 0.3

While not wishing to be bound by any particular theory of the invention,cold storage not only delays senescence but, as discussed above, induceslasting physiological changes that cause an increased embryogenic growthrate after recovery at normal growth temperature. The enhanced growthrate has been maintained at least for 2-3 months under prolonged coldconditions. Viability was retained at 100% in the cold (at 5-10° C.) forat least 10 months with testing every 4 weeks. Recovery from coldstorage (and totipotency) can be monitored by transferring the culturesinto differentiation inducing conditions (secondary medium under lightand culture room conditions (25-27° C.)) and measuring the changes ofchlorophyll content (a good indicator of the amount of shootdifferentiation in this system) as well as the growth rate.

IT is noted that the present inventors initiated the first embryogenicculture of Arundo donax in 1998 and this culture has been maintainedwithout loss of regeneration ability for more than 8 years. The methodsof the present invention have also allowed the initiation of embryogeniccell cultures consistently from this same wild clone of Arundo and fromother ecotype clones from different habitats routinely every year.Several lines have been developed from the “Blossom”, and “GT” ecotypes.To the best of the inventors' knowledge, the present invention is thefirst report of sustained totipotent embryogenic cell culture of Arundodonax.

The observed positive effects of cold treatment on the cultures weresurprising, because Arundo donax as well as some of the other species ofthe present invention are Mediterranean or subtropical species and thus,are considered to be cold sensitive.

Example 2 Productivity and Propagation Yield

The following is an exemplary experiment showing the productivity andthe propagation yield of the present invention.

1. Culture dish multiplication (primary and secondary cultures). A fiveto ten times multiplication rate per four weeks was achieved in theprimary and secondary cultivation steps (0.6 g to 1 g→6 g to 7.5 g per 4week cycle). Approximately 200,000 to about 3,200,000 potential plantswere transferred per hour (considering one to four tertiarysubcultures). About 10 to 20 million potential plants were produced perm² embryogenic culture room floor space (considering a single tertiarysubculture and 4000 dishes per m³ box).

2. Transfer to culture vessels (tertiary culture) and multiplication.About a four times multiplication rate per 3 week cycle (30 g→120 g) wasachieved with about a 5% attrition rate. Thus, about 240,000 potentialplants were transferred per hour.

The present invention resulted in a high volume output. Thus, from asingle culture dish of embryogenic cell culture (6-7.5 g) after 12 weeks(four tertiary subcultures) 160,000 potential plants entered into theacclimatization process.

3. Quaternary culture: acclimatization (two to four weeks). Step 3included plants ready for transfer to greenhouse flats as well as fourweek-old field ready plants.

Parameters used in the calculations include the following. Eachfully-grown culture dish provides about 6 g to 7.5 g embryogenicculture, which is approximately 2,500 potential plants. The tertiarycultures are initiated using about 30 g of embryogenic tissue per vesselor four culture dishes of tissue per vessel. The fresh weight of a fullygrown culture in one vessel is 120 g which is approximately 10,000potential plants.

Example 3 Transformation of Arundo donax Explants Using Agrobacteriumtuinefasciens

Agrobacterium tumefaciens carrying the plasmid, pMSF3022, was used totransform Arundo donax explants (cross-sectional segments of immatureinflorescence). Plasmid pMSF3022 carries the bar gene that confersresistance to phosphinothricin and provides a positive selection and thegfp (green fluorescent protein) gene which provides a visual selectionof transformants. Antibiotic resistant transformants were selected forfour weeks after gene transfer by incubating the explants on primarysolid culture medium with or without phosphinothricin and containingticarcillin at 400 m/L concentration to eliminate residualAgrobacterium. As shown in FIG. 6A control explants (untransformed) werekilled by 10 mg/L of phosphinothricin (antibiotic/herbicide). In theabsence of the antibiotic/herbicide, the control explants developembryogenic callus (FIG. 6B). In contrast, the explants that werecocultivated with the A. tumefaciens carrying the plasmid, pMSF3022(transformed), were able to develop embryogenic callus in the presenceof 10 mg/L of phosphinothricin (FIG. 6C). FIG. 6D shows thatcocultivated explants develop embryogenic callus in the absence of theantibiotic/herbicide.

Example 4 Somaclonal Variation in the Sustained Totipotent EmbryogenicCell Cultures

Arundo donax plants regenerated from the embryogenic cell cultures grownin liquid secondary growth medium as described herein show higherfrequency of somaclonal variation than vegetative clones produced bycutting in the greenhouse. The trait chosen to demonstrate somaclonalvariation is oxidative dehalogenation activity of cell-free extract ofroots. The mean dehalogenation activity of the population was shown toincrease in the embryogenic cell cultures grown in liquid secondarygrowth medium as compared to vegetative clones produced by cutting inthe greenhouse.

1. Assay for oxidative dehalogenation activity. The colorimetric assayto measure oxidative dehalogenation measures peroxide-dependentenzymatic dehalogenation of 2,4,6-trichlorophenol (TCP) by cell-freeextract of roots. TCP is an anthropogenic environmental pollutant, whichis toxic to cell cultures and plants. Oxidative dehalogenation activityis proportional to total root peroxidase activity assayed by TCP, whichconveniently acts as the chromogen in the reaction.

Roots are separated, rinsed with distilled water, blotted dry, and, ifnot used immediately, frozen in liquid nitrogen for temporary storage.Samples are weighed, frozen in dry ice-ethanol bath and homogenized inthe extraction buffer (50 mM KPO₄ buffer, pH 5.3) in a microcentrifugetube with a micro pestle. The homogenate is centrifuged at 15,000×g for20 min.

For a standard enzyme assay, 500 μliter of the supernatant (crudeextract) is added to 500 μliters of assay buffer. The assay buffercontains 20 mM H₂O₂, and 1 mM TCP in 20 mM Bistris buffer (pH 5.9, 25°C., 1 hr). The intensity of the color at 525 nm is proportional to theloss of TCP as measured by HPLC.

TCP removal from the liquid phase is assayed by measuring TCP on a3.8×150 mm NovaPak-C18 reverse phase column (Part No. WAT086344, Waters2690 HPLC separations module with Waters 996 photodiode array detector,Millenium32 Operating System, Waters Co.) under isocratic conditions(sample size, 50 μliters; flow rate, 1 ml/min, mobile system, acidicmethanol (MeOH-5% acetic acid in water, 70:30 detection is at 289 nm)).

2. Preselection of the embryogenic cell cultures on a halogenatedxenobiotic results in increased somaclonal variation. In order topreselect the embryogenic cell cultures, TCP is dissolved in methanol,filter sterilized and added to the autoclaved cooled medium just beforesolidification under aseptic conditions. Browning of the tissue occursafter three days on TCP concentrations of 0.3 mM or greater. The minimumconcentration at which necrosis and growth arrest occurs is 0.24 mM.Partial browning occurs between 0.24 and 0.28 mM TCP, but some tissuesurvives even at this concentration. Preselection is carried out bygrowing embryogenic cell cultures on 0.24 mM TCP. Enzyme activity isassayed 2 months later (see above).

The application of a selection pressure alters the frequency ofsomaclonal variants. Exposure to near lethal levels of TCP results ingreater genetic variability as assayed by dehalogenation activity. FIG.7 demonstrates the dispersion of the genetic variability resulting fromthese experiments. The activity, standardized to fresh weight, issubdivided into arbitrary 1-unit ranges and the relative number (% oftotal tested) of individuals falling into the range is indicated on theY axis of FIGS. 7A, 7B and 7C. Compared to nursery propagation bycuttings as shown in FIG. 7A, the embryogenic cell culture basedpropagation shown in FIG. 7B resulted in a 14% increase in the meandehalogenation activity of the population above the 95^(th) percentile(top 5%). For the top 1%, the increase was 31%.

Additionally, exposure of the embryogenic cultures to near lethal levelsof TCP resulted in greater genetic variation in the final plantpopulation as assayed by dehalogenation activity and shown in FIG. 7C.FIG. 7C shows the increase of mean activity of the population above the95^(th) percentile (top 5%) was 64% as compared to vegetativepropagation. For the top 1%, the increase was 93%. FIG. 7D also presentsthe data charted as quartiles.

Example 5 Prescreen for Compatible Versus Incompatible Plant-MicrobeAssociations

In order to determine which plant-microbe associations are compatibleand which are not, prescreening of the various associations can beundertaken.

For this prescreening assay, excised sterile Arundo donax roots aredipped into liquid cultures of individual strains of bacteria and theroots are placed in quaternary plant culture medium, liquid orsolidified with a gelling agent. Non-compatible bacteria causediscoloration, infiltration, hypersensitive reactions and finallynecrosis of the roots. Compatible strains in which the roots remainnormal are re-tested with intact plants.

Example 6 Oil Degradation in Plant-Microbe Associations as Detected by aMicrofilm Degradation Assay

Disappearance of crude oil residue and select polyaromatic hydrocarbonsin contact with bacteria-colonized roots can be visualized by amicrofilm degradation assay. A thin film of crude oil is dried on thesurface of minimal nutrient medium without carbon source by evaporationfrom a nonpolar solvent (for example, hexane) solution. The contrastbetween the oil film and the region cleared up by the bacteria (showingup as opaque) colonizing the roots is visualized by the photographictechnique, which produces an opaque (whitish) appearance due to anopaque paper showing through the clear window created by the digestionof the oil film. FIG. 8 shows an example of a microfilm degradationassay in which Arundo donax roots are colonized by Pseudomonas cepaica.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1-54. (canceled)
 55. A method of producing and maintaining a sustainedtotipotent embryogenic cell culture of a monocotyledonous plantcomprising: (a) cultivating an explant of tissue from a monocotyledonousplant to produce a sustained totipotent embryogenic cell culture,wherein the monocotyledous plant is selected from the group consistingof Scirpus spp. and Thysanolaena spp.; (b) treating the sustainedtotipotent embryonic cell culture with a cold temperature, wherein thecold temperature treatment is for a duration from about 60 days to about360 days and comprises a temperature in a range from about 4° C. toabout 10° C.; (c) removing the sustained totipotent embryogenic cellculture from the cold temperature treatment; and (d) cultivating thesustained totipotent embryogenic cell culture, whereby the sustainedtotipotent embryogenic cell culture of the monocotyledonous plant has anincreased growth rate as compared to a control.
 56. The method of claim55, further comprising regenerating a plantlet and/or plant with rootsand shoots from the sustained totipotent embryogenic cell culture ofstep (d).
 57. The method of claim 56, comprising transferring theplantlet and/or plant to a medium without hormones or directly to soilfor acclimatization to non-sterile and photosynthetic conditions andproducing an acclimatized plantlet and/or plant.
 58. The method of claim55, wherein the totipotent embryogenic cell culture of step (d) isstored, transported and rehabilitated for the production of propagules.59. The method of claim 55, wherein the sustained totipotent embryogeniccell culture is stored long-term at a cold temperature, with or withoutsubculturing of the sustained totipotent embryogenic cell culture. 60.The method of claim 55, wherein the explant is from a transgenic plantstably transformed with at least one heterologous nucleotide sequence.61. The method of claim 55, comprising introducing at least oneheterologous nucleotide sequence into a cell of the sustained totipotentembryogenic cell culture to produce a transgenic sustained totipotentembryogenic cell culture.
 62. The method of claim 61, comprisingproducing a transgenic plantlet and/or plant from the transgenicsustained totipotent embryogenic cell culture.
 63. A method ofestablishing a plant-microbe association, comprising co-cultivating atleast one plant produced by the method of claim 56 with at least onemicrobial species in a medium without hormones, thereby establishing aplant-microbe association.
 64. The method of claim 63, comprisingacclimatizing the plant having an established plant-microbe associationto non-sterile and photosynthetic conditions.
 65. The method accordingto claim 56, further comprising using the plantlet and/or plant inphytoremediation, phytoreactor systems or biomass production.
 66. Themethod according to claim 57, further comprising using the plantletand/or plant in phytoremediation, phytoreactor systems or biomassproduction.
 67. The method according to claim 62, further comprisingusing the transgenic plantlet and/or plant in phytoremediation,phytoreactor systems or biomass production.
 68. A method ofphytoremediation, comprising: establishing a plurality of the plants ofclaim 56; and contacting the roots of the plants with an environmentalpollutant in a liquid medium and/or a land area, thereby causing theenvironmental pollutant to be removed from the liquid medium and/or theland area.
 69. A method of phytoremediation, comprising: establishing aplurality of the plants of claim 57; and contacting the roots of theplants with an environmental pollutant in a liquid medium and/or a landarea, thereby causing the environmental pollutant to be removed from theliquid medium and/or the land area.
 70. A method of phytoremediation,comprising: establishing a plurality of the plants of claim 62; andcontacting the roots of the plants with an environmental pollutant in aliquid medium and/or a land area, thereby causing the environmentalpollutant to be removed from the liquid medium and/or the land area. 71.A method of phytoremediation, comprising: establishing a plurality ofthe plants of claim 64; and contacting the roots of the plants with anenvironmental pollutant in a liquid medium and/or a land area, therebycausing the environmental pollutant to be removed from the liquid mediumand/or the land area.
 72. A method of producing an elite plant line,comprising: selecting for at least one trait of interest in thesustained totipotent embryogenic cell culture of claim 55, wherein theat least one trait of interest is a result of somaclonal variation inthe embryogenic cell culture or the introduction of at least oneheterologous nucleotide sequence into the genome of a cell of theembryogenic cell culture; and cultivating the sustained totipotentembryogenic cell culture comprising the at least one trait of interest,thereby producing an elite plant line.
 73. The method of claim 72,further comprising regenerating a plantlet and/or plant with roots andshoots from the sustained totipotent embryogenic cell culture.
 74. Amethod of producing an elite plant line comprising: selecting for atleast one trait of interest in the plantlet and/or plant of claim 56,wherein the at least one trait of interest is a result of somaclonalvariation or the introduction of at least one heterologous nucleotidesequence into the genome of a cell of the plantlet and/or plant; andcultivating the plantlet and/or plant comprising the at least one traitof interest, thereby producing an elite plant line.
 75. The methodaccording to claim 72, further comprising using the plantlet and/orplant in phytoremediation, phytoreactor systems or biomass production.76. The method according to claim 74, further comprising using theplantlet and/or plant in phytoremediation, phytoreactor systems orbiomass production.
 77. A method of phytoremediation comprising: (a)establishing a plurality of the plants of claim 73; and (b) contactingthe roots of the plants with an environmental pollutant in liquid mediumand/or a land area, thereby causing the environmental pollutant to beremoved from the liquid medium and/or the land area.
 78. A method ofphytoremediation comprising: (a) establishing a plurality of the plantsof the elite plant line of claim 74; and (b) contacting the roots of theplants with an environmental pollutant in liquid medium and/or a landarea, thereby causing the environmental pollutant to be removed from theliquid medium and/or the land area.
 79. A sustained totipotentembryogenic cell culture of a monocot plant that is produced by themethod of claim
 55. 80. A transgenic sustained totipotent embryogeniccell culture of a monocot plant that is produced by the method of claim60.